1. Field of the Invention
The invention relates to the field of micro-analytical chemistry in the areas of cellular biochemical and biomedical analysis, and in particular to a class of chemical compounds and method and apparatus for assaying the activity of oncoproteins in a single cell or selected cells.
2. Description of the Prior Art
In biology, the individual cell can be thought of as the fundamental unit of life. phenomena that we observe in living organisms. Most intracellular chemical processes are mediated by proteins. Typically these proteins are enzymes that catalytically enhance the rates of specific chemical reactions. Very often, intracellular enzymes participate in cascades of chemical reactions known as signal transduction pathways. Each of these signal transduction pathways is composed of sequentially acting enzymes, frequently of a class known as kinases. These enzymes not only interact with and modify the behavior of other proteins within the same pathway, but also influence the operation of other signal transduction pathways. These interacting cascades of signaling molecules and chemical reaction products form complex networks that ultimately regulate processes of cell growth, proliferation, quiescence, and programmed cell death.
Inappropriate signaling within a cell can give rise to defects in any of these processes and is implicated as the basis for many forms of cancer. To further complicate matters, individual cells display a high degree of heterogeneity in their internal biochemical signaling. Malignant tumors are typically composed of such a heterogeneous group of cells, and cells within the same tumor often utilize disparate and aberrant growth signaling pathways. This heterogeneity makes it necessary to analyze individual cells to elucidate their unique errors in signaling. Where only a small proportion of cells show aberrant signaling, analysis of an entire population will produce an average signal more reflective of the majority rather than of any individual aberrant cell. Thus, the measurement xe2x80x9caverages outxe2x80x9d the very errors one seeks to detect.
Often there is a genetic basis, in the form of mutations in genes for signaling proteins, for inappropriate signaling. Mutations in the genes for signaling proteins result in the presence of structurally altered, aberrantly acting, signaling proteins. Currently, there exist methods to analyze the DNA and/or RNA of a single cell to detect the presence of signaling protein mutations and thereby infer the presence of mutant signaling proteins. However, it is not currently possible to predict or understand errors in signaling purely from knowledge of the genes or mutations in the genes that occur within a tumor. For example, the aberrant activity of mutant signaling proteins in one pathway may be counteracted or modified by the activities of other proteins, mutant or normal, involved in other pathways. This is very likely since the interior of a tumor cell is a complex mixture of signaling proteins generally with hundreds to thousands of different kinds present simultaneously and participating in pathways that frequently influence one another. To assess the function of any single kind of a signaling protein, its net activity must be measured from within a single living cell.
Further, to completely describe the function of an entire pathway, measurements of the activities of all the signaling proteins involved within a pathway are needed. Such knowledge would be exceptionally useful in the individualized diagnosis and treatment of diseases that involve faulty intracellular signaling since signaling proteins are often prime targets for therapeutic drug intervention.
Further, such a capability would revolutionize the ability to conduct research on basic cellular physiology. As noted, kinases represent an exemplary class of protein enzymes that commonly play a key role in intracellular signaling. In fact, aberrant activation of growth promoting kinases is a general feature of tumor cells. Thus protein kinases are promising targets for cancer therapies and the development of kinase antagonist drugs is an area of intense research. These enzymes catalytically enhance the rate at which a covalent chemical bond is formed between a phosphate group and a molecular substrate molecule, frequently, a different protein. This process is termed xe2x80x9cphosphorylationxe2x80x9d. When the substrate itself is an enzyme, such phosphorylation generally either enhances or suppresses the substrate enzyme""s chemical activity.
Kinases generally demonstrate substrate specificity such that the preferred substrates of one kind of kinase are not efficiently phosphorylated by other kinds of kinases. Frequently, the preferred substrates for kinases are different kinases; thus, the inappropriate activity of one kinase can result in changes in the activity of multiple downstream kinases within a signaling pathway.
A general method to measure kinase activity within a cell could, in many instances, yield an abundance of information about an entire signaling pathway. However, in order to make such a measurement strict criteria must be met. To begin with, the number of copies per cell of many enzymatic proteins, such as kinases and kinase substrates, can be as low as 100 to 1,000,000 molecules, or about 150 pM to 1.5 xcexcM in concentration in a typical mammalian cell with a volume of 1 pl. In terms of moles, this is equivalent to a necessary limit range of approximately 1.7xc3x9710xe2x88x9218 to 1.7xc3x9710xe2x88x9222.
Further, since the concentrations of phosphorylated substrates in cells change on time scales of the order of seconds, the time resolution of the measurement, from the instant the contents of a cell are obtained to the time that the biochemical reactions are terminated, must be sub-second. Most conventional biochemical assays meet neither the temporal resolution nor the sensitivity limits required for these single cell measurements. The temporal resolution requirement can be met through the use of apparatus described in application Ser. No. 09/036,706 filed Mar. 6, 1998, now U.S. Pat. No. 6,156,576, and entitled, xe2x80x9cFast Controllable Laser Lysis of Cells for Analysisxe2x80x9d, to which this continuation-in-part application is related and which parent application is herein expressly incorporated by reference. The necessary degree of sensitivity can be achieved with traditional capillary electrophoresis (CE) methods. Lacking, until now, has been the molecular means to accurately determine the intracellular activity of one or more kinase species or other enzymes.
Current techniques for kinase measurements can generally be divided into three methodologies. The first method uses the phosphorylation of kinase substrates by cellular extracts to estimate the kinase activity that occurs within intact cells There are a number of major drawbacks to such a method. Because it is not sensitive, the internal contents of large numbers of cells must be pooled. Since the cells are not synchronous with respect to their activation status, a time averaged level of kinase activity is actually measured. Furthermore, during the time required to generate a cellular extract, a time that may span many seconds to minutes, many chemical reactions continue to proceed. This results in a highly distorted representation of the relative amounts of reactants and products as they occurred within actual cells.
Even beyond these difficulties, it is virtually impossible to reproduce the unique chemical microenvironments that occur within cells; thus, the chemical activity observed in extracts can differ greatly from that which actually occurs within intact cells.
A second methodology relies on labeling the kinase within a cell with specific antibodies, kinase inhibitors, or fluorescent tags and then observing the cells with a fluorescence microscope. Both inactive and active kinase molecules are labeled by this method, and an attempt is then made to infer the activity level of the kinase from its actual location within a cell. However, it is extremely risky to attempt to determine the state of activation of an enzyme solely from its intracellular location. This is particularly true for a number of kinases that are active both when free in the cell and also when bound to intracellular structures.
A third method, still under development, is the use of a fluorescent indicator to actually measure kinase activity. A similar strategy has worked well for the measurement of various intracellular ion concentrations (i.e. Ca2+), but thus far has not been generally applicable to the measurement of kinase activities. One of the drawbacks of this approach is the need for a supraphysiologic concentration of a reporter fluorophore to produce sufficient signal for ready detection-typically 10 xcexcM to 100 xcexcM of fluorescent indicators are necessary within cells for detection by fluorescence microscopy. This is 10 to 1,000,000 times the concentration of typical physiological kinase substrates. At such high concentrations the fluorescent reporter molecule may behave as an inhibitor or may induce other unintended cellular responses such as activation of aberrant signaling pathways. Another difficulty has been that the viscous, highly concentrated, intracellular environment alters the fluorescent properties of the reporter molecule making the detection unreliable.
Finally, the design of fluorescent, environment-sensitive probes for a specific kinase may not be generally applicable to other kinases. Because a given cell or group of cells may contain a large number of different kinases, this may pose a serious problem. The need for broadly applicable, accurate, and sensitive cellular measurement technologies is great.
What is needed is an alternative, yet complementary, strategy to the above approaches. Further, this new approach should be one that it is not limited to the measurement of kinases, but is broadly adaptable to the measurement of many if not all other kinds of intracellular enzymatic and chemical activities.
A biomedical arena in which the LMS assay system will prove of immense scientific and commercial value is cancer biology, including molecular diagnosis and drug discovery. The contemporary view of cancer suggests that it arises from molecular defects in single cells. These defects result in the excess activity of components comprising the signal transduction networks involved in cell proliferation. Of particular import are enzymes in the networks especially the kinases. In fact of the approximately 120 known oncogenes, the majority encode for proteins which are kinases. With somewhere between 2000 to 4000 kinases, the function of many which remains unknown, it is probable that the role of such enzymes in cancer biology will take on greater importance.
At present the understanding of the molecular mechanisms underlying carcinogenesis remains rudimentary. However, there is one cancer, chronic myelogenous leukemia (CML), that has been well-characterized in terms of the critical error that leads to malignancy. CML accounts for about 15 percent of all leukemias in adults and strikes most often between the ages of 45-65 years. With the increase in the number of xe2x80x9cbaby-boomersxe2x80x9d reaching this age bracket, the incidence of this disease can be expected to increase over the next few decades.
In the 1960""s analysis of chromosomes in patients with CML showed the presence of an abnormal chromosome called the Philadelphia chromosome. Since that time it has been found that the abnormal chromosome is due to the breakage of a piece of DNA off of one chromosome and its reattachment to another. This results in the fusion of two genes, xe2x80x9cbcrxe2x80x9d and xe2x80x9cablxe2x80x9d. The abl gene encodes a tyrosine kinase, and the fusion of the abl and bcr genes produces an oncogene encoding a protein with increased and unregulated kinase activity. If this bcr-abl oncogene or its corresponding protein is put in a normal cell, that cell is transformed into a cancerous cell. It is now known that the constant activation of the kinase turns on signaling pathways in the cell that leads to the cell""s unregulated proliferation, or cancerous state. This paradigm is a model for the molecular etiology of other cancers, and a great deal of effort is ongoing to define such errors in almost all cancers. It can be expected that the protein products of most oncogenes play important roles in their associated cancers. Oncogenes encoding tyrosine kinases are present in many cancers including cancers of the breast and prostate, two of the most common types of cancer.
This information on molecular abnormalities is currently being used in the treatment of cancer. The diagnosis and the differentiation of CML from other blood diseases rely on molecular studies for the presence of the Philadelphia chromosome, the bcr-abl oncogene and the abnormal bcr-abl enzyme. Such studies as metaphase analysis, Southern blot, fluorescence in situ hybridization, western blot, and PCR are all used routinely for CML, and provide valuable information for the physician. However, these studies all require a significant amount of manpower, time and technical skill. These tests take anywhere from two days to two weeks to perform.
The LMS assay provides unique information and offers distinct advantages over these techniques. The LMS assay measures the activity of the abl tyrosine kinase in the intact cell. Perhaps of greater import is that the assay can literally be done in less than an hour and requires less manpower to perform than current methods.
In the area of drug discovery pharmaceutical companies have a strong interest in developing drugs that block the specific enzymes and pathways that drive the cellular proliferation characteristic of all cancers. In the case of CML, it is clear that the bcr-abl protein is a target for drug development, and tyrosine kinase inhibitors are aimed for clinical trials. Current assays to screen for inhibition of tyrosine kinases require significant investments in manpower, are performed in a nonphysiologic manner, or do not perform direct measurements on the molecular target of interest.
The LMS assay for abl activity provides new and valuable information with less effort, time, and costs than current methods greatly benefiting the pharmaceutical industry.
The present invention is a method for measuring intracellular chemical activity relating to detection and drug inhibition testing of oncogenic proteins. Typically the molecules of interest are intracellular enzymes, proteins that catalyze biochemical reactions within cells, or more specifically kinases. Catalysis by enzymes results in the alteration of the chemical structure of substrate molecules. The invention comprises the use of substrate molecules that undergo a change in electrophoretic mobility when enzymatically acted upon as intracellular reporters of chemical activity. This strategy provides a general method to detect and quantify enzymatic activity by measuring changes in the structure of substrate molecules which are associated with cancerous cellular microbiology. A change in electrophoretic mobility allows for separation of enzymatically altered substrate molecules related to oncogenic cellular activity from unaltered substrate molecules by electrophoresis with highly sensitive detection and quantification methods following separation. Even very minor changes in chemical structure can result in measurable differences in electrophoretic mobility. For example, capillary electrophoresis is routinely used to separate stereoisomeric forms of chemical compounds.
Most commonly, substrate molecules are selected to report on the activity of a specific enzyme or class of enzymes. These reporter substrate molecules can occur naturally within a cell, be induced within a cell, such as when under the control of an operon or similar genetic control mechanism, or be introduced into a cell by a variety of established methods including, but not limited to, microinjection, electroporation, optoporation, vesicle fusion, pinocytic loading, or by association with membrane permeant peptides. The reporter substrate molecules may be naturally occurring compounds or synthetically derived. Measurements are made with the intracellular presence of such substrate molecules, at some time of interest, typically after exposure of a cell to a stimulus, such as the onset of cancerous bioactivity. At the time of interest, part or all of the contents of a single cell or group of cells are harvested for separation by capillary electrophoresis. To ensure accuracy of measurement, this process must be achieved in a timely manner so as to minimize chemical reactions occurring subsequent to the time of interest. In practice, this means that if a measurement is to be made to within an accuracy of 1%, then less time should pass, from the time of interest to the time of termination of chemical reactions, than is required for the progress of a reaction to change by 1%. Typically, chemical reactions are terminated by physical disruption as with detergents, turbulent mixing, dilution, or separation of molecules by electrophoresis, or by a combination of these processes. Fast controllable laser lysis is an excellent way to harvest, on a sub-second time scale, the contents of a single cell into which reporter substrate molecules have been introduced.
Reporter molecules specific for particular intracellular reactions can be designed to undergo a predictable change in electrophoretic mobility. These molecules can also be modified to be fluorescent and thus detectable in the minute amounts and low concentrations that occur within the tiny volumes of single cells. In the illustrated embodiments, modified peptides are used as representative reporter molecules. Modified peptides are a particularly useful class of such possible reporter molecules for a number of reasons. Foremost, peptides closely mimic natural, endogenous protein substrates and frequently demonstrate a high degree of specificity in the reactions in which they participate and thus report. This can be understood to be the result, in many cases, of the unique primary sequence structure of these peptides. The particular order of amino acid residues that comprises a given peptide conveys unique chemical properties to the molecule, particularly with regards to being recognized and acted upon as a substrate by a particular enzyme. Typically, enzymes whose natural substrates are proteins will efficiently catalyze only those reactions involving molecules that display a particular order or pattern of amino acid residues.
Peptides are extremely versatile compounds when used as reporter substrates. Large, diverse libraries of these compounds can be generated simply by varying the sequence and number of amino acid residues that comprise a given peptide. Furthermore, modifications of basic peptide structure can be made in a number of ways. For example, D-isomer amino acids can be incorporated into peptides in place of the more commonly found L-isomeric forms. Also, unusual or alternate, non-naturally occurring amino acids can be incorporated into peptides. Examples include homo-arginine and homo-lysine in place of arginine and lysine, respectively. Even the backbone peptide bond may be altered. After modifications such as these have been made, the resultant peptides have proven to serve as substrates for enzymatic reactions.
In the illustrated embodiments, the modified peptides are substrates for enzymes known as kinases that alter the peptides by the addition of a phosphate moiety to a specific amino acid(s) within the peptide. Phosphorylation can be effected in vitro, in the absence of a cell, and leads to a demonstrable alteration of the peptides"" chemical structure and electrophoretic mobility.
Further, the peptides used in the illustrated embodiments are modified at at least one point, frequently the end by covalent addition of a fluorescein group (or other fluorescent group) to allow detection by laser-induced fluorescence. The fluorescent tags can be added to either the amino or carboxyl termini or to specific amino acid residues by chemical techniques well-known to those of skill in the art. Examples of readily modifiable residues include the sulfhydryl group of cysteine, the amino groups of arginine and lysine, and the carboxyl groups of aspartate and glutamate. Example chemical groups that react with sulfhydryl groups include maleimides, iodoacetamides, alkyl halides, aziridines, and epoxides. Chemical groups that react with amines include succinimydyl esters, sulfonyl halides, isothiocyanates, and aldehydes. Chemical groups that react with carboxyls include carbodiimides, hydrazines, alkyl halides, amines, and diazoalkanes. A wealth of different fluorophores, with reactive groups, are commercially available for covalent linkage to peptides at these groups. Examples of different fluorophores that are available include fluorescein derivatives such as Oregon Green produced by Molecular Probes (Eugene, Oreg.), rhodamine derivatives such as Texas Red and tetramethylrhodamine, NBD (7-nitrobenz-2-oxa-1,3, diazole) derivatives, coumarins, dansyl derivatives, pyrenes, and the cyanine dyes (Cy-3/Cy-5) produced by Amersham/Pharmacia Biotech (Piscataway, N.J.). This diversity allows the preparation of peptides that absorb and emit light at wavelengths ranging from the ultraviolet through the visible and into the infrared parts of the spectrum. This is yet another avenue that can be exploited to produce a large variety of reporter substrate peptides with differing spectral properties.
While illustrative, the invention is not contemplated to be limited to use of modified peptides alone or in its scope to the monitoring of reactions by intracellular kinases alone. It is contemplated that a broader range of intracellular chemical reactions can be monitored by this approach. For example, peptide-based reporter substrates may be designed to report on the activities of phosphatases, glycosylases, acetylases, proteases and caspases, and isomerases. It is also contemplated that molecular reporters may be based on other chemical species commonly found in cells such as nucleic acids, carbohydrates, phospholipids, and entire proteins or may be based on compounds (often polymers) not ordinarily found within cells. Nucleic-acid based reporter molecules could serve as excellent substrates for nucleases, ligases, polymerases, methylases, demethylases, and nucleotide transferases. Lipid based reporter substrates could serve to monitor lipases. Carbohydrate based reporters could serve to monitor oxidoreductases, glycosylases, hydrolases, lyases, and isomerases. Finally since entire proteins are frequently the natural substrates for intracellular chemical reactions, reporters based on these could prove to yield extremely accurate information about intracellular physiology.
Reporter molecules that comprise entire proteins may be based on the green fluorescent protein (GFP) from the jellyfish Aequorea victoria. This protein is intrinsically fluorescent and has been used extensively in basic biological research. The gene for the protein has been cloned and mutated to produce a diversity of GFP-like fluorescent proteins that have unique absorption and emission spectra. A gene for a GFP-like protein can be linked to genetic sequences for either a peptide or an entire protein. Upon expression within a cell, such a chimeric molecule has the fluorescent properties of GFP along with the properties of the linked peptide or protein. When the linked peptide or protein is a substrate, the result is a protein-based fluorescent molecular substrate. Such a chimeric molecule can be produced in bacteria or cultured eukaryotic cells and purified for use as an exogenous reporter substrate, or the gene can be introduced into the cell of interest for the endogenous production of reporter substrate. In fact, such a gene can be introduced into a cell-line to produce a new cell-line that carries the gene from one generation to the next. After intracellular turnover and sub-cellular sampling, electrophoresis can be used to separate the GFP-linked turned-over substrate molecules from non-turned over GFP-linked substrate molecules. It is clear that genes for fluorescent proteins exist in other organisms, such as Renilla, that are not identical to the GFP from Aequorea, and it is expected that when these genes are cloned they will be similarly utilized.
Detection of molecular reporter molecules, obtained from within a cell, need not be achieved only by fluorescent methods. Fluorescent methods are generally utilized because of the extremely low limits of detection that they provide. In fact, fluorescence-based methods have been developed that can allow the detection of single molecules. As illustrated in Table 1, the necessary detection limit to observe the amount of substrate found in a 1 pl (picoliter) mammalian cell at a physiologically normal concentration of 1 xcexcM, is 10xe2x88x9218 mol or about 600,000 molecules. Alternative quantitative optical spectroscopic detection methods based on phosphorescence or chemiluminescence can also achieve this low limit of detection. Also some electroanalytical methods such as amperometry or voltammetry are sensitive enough to be used to detect and quantify altered and unaltered reporter molecules obtained from single cells. Sensitive detection and quantitation has also been achieved by coupling electron ion-spray mass spectrometry to capillary electrophoretic separation. Incorporation of heavy atoms into the reporter molecules, either from elements rare to biological systems or in the form of unusual isotopes, could greatly increase the utility of such mass spectrometry.
Multiple reporter substrates, each reporting on a specific chemical reaction, are expected to be utilizable simultaneously within a given cell.
Finally, it is anticipated that the use of reporter molecules, as disclosed herein, will have utility in contexts that are not exclusively intracellular. This approach will be generally applicable to the measurement of chemical activities in circumstances where minute volumes, low concentrations, and miniscule quantities occur. An example of a non-cellular volume that could be assayed with such reporter molecules is the interior of a liposome or vesicle, a single-cell sized, or smaller, compartment that is typically enclosed by lipid molecules.
The invention and its various embodiments, now having been summarized, may be better understood by viewing the following drawings wherein like elements are referenced by like numerals.